Landscape of TET2 Mutations In Acute Myeloid Leukemia (AML): A Next-Generation Sequencing Study Investigating 76 Cases Comprehensively Characterized for Cytogenetics and Other Molecular Markers.

Abstract 1035

Mutations of the ten eleven translocation (TET2) gene have been reported to be frequent in hematological malignancies. However, data are preliminary and investigation is challenging and labor-intensive with standard sequencing techniques. Here, we used massively parallel Titanium amplicon next-generation sequencing (NGS) technology (454 Life Sciences, Branford, CT) and investigated 76 patients with acute myeloid leukemia (AML), including 66 de novo AML, 6 s-AML and 4 t-AML cases, respectively, diagnosed between 8/2005 and 5/2010. The median age of the cohort was 64.7 years. According to cytogenetically defined MRC criteria (Grimwade et al., Blood 2010) 63 patients of our cohort were assigned to the intermediate prognostic risk group and 13 to the poor risk group. Patients of the favorable prognostic risk group or those with “recurrent genetic abnormalities” according to WHO classification were excluded. In detail, 61 patients had a normal karyotype, 4 had other chromosomal aberrations and 11 had a complex aberrant karyotype. All coding exons of TET2, represented by 27 distinct PCR amplicons, were examined. For each amplicon a median of 643 reads was generated, thereby allowing a sensitive detection of variants, i.e. at a cut-off value of 10% 64 bidirectional reads were generated. In total, we observed 56 variances by this molecular mutation screening. After excluding 13 different polymorphisms and 1 silent mutation, 42 distinct mutations were detected in 26/76 (34%) patients. We identified 23 point mutations (14 missense and 9 nonsense; 55%) and 18 frameshift mutations (12 deletions, 5 duplications and 1 insertion; 43%). In 1/76 (2%) patients a splice site mutation was identified. The frameshift mutations ranged from 1 bp to 8 bp for deletions and 1 bp to 52 bp for duplications/insertions. The observed TET2 mutations were found to be heterogeneous and were spread over several exons. However, exons 3 and 11 could be identified as mutational hotspot regions, where 30/42 (71%) of the mutations were located. In exons 4, 5 and 9 only one mutation each was observed, respectively. No mutation was detected in exon 8. In detail, 25/42 (60%) mutations were located outside of the two conserved regions as described by Delhommeau et al. (N Engl J Med 2009), whereas 13 mutations (31%) were observed within the first conserved region spanning from codons 1134 to 1444 and 4 mutations (9%) were found within the second conserved region covering codons 1842 to 1921, respectively. 11/13 of the variances in the conserved regions were nonsense or frameshift mutations. Of the observed 42 mutations, only 8 had previously been described in the literature. The other 36 mutations represented here are novel. Generally, our results extend data known from the literature, i.e., the frequency of 34% of TET2 mutations in our AML cohort is higher than in previous publications, reporting a frequency between 12% and 20%. Concerning the mutational burden, the median range of the identified mutations was 44% of sequence reads carrying the mutation. Mutation load <20% was only observed in 2 cases (10% and 11%, respectively) and therefore would be below the detection level of Sanger sequencing. None of the detected TET2 mutations had a mutational burden of <10%. 15/26 (58%) patients carried more than one mutation and a mean of 1.6 mutations per patient was observed. In 2 patients two mutations within the same amplicon were detected and NGS was able to delineate subclones, i.e. in both cases approximately half of the reads carried either one mutation and only 1.7% and 2.3% of reads, respectively, harbored both mutations concomitantly. We further characterized this cohort according to mutations in NPM1 (n=73 cases investigated), FLT3-ITD (n=74), FLT3-TKD (n=64), CBL (n=76), and IDH1 (n=49). TET2 mutations were concomitantly observed with mutations in other molecular markers such as NPM1, FLT3-ITD, FLT3-TKD or CBL. Surprisingly, we found that TET2 mutations significantly excluded mutations in IDH1 (p=0.004). With respect to clinical data no difference in overall survival was monitored for AML patients that carried TET2 mutations (TET2-mutated 347 days vs. TET2 wild-type 294 days, n.s.). In conclusion, TET2 is a frequently mutated gene in AML. Due to the increasing complexity of observed molecular aberrations future studies will be essential to clarify the clinical relevance of TET2 mutations in AML and its prognostic and therapeutic role.